Nucleotide and nucleic acid biosynthesis is fundamental to both the uncontrolled proliferation of cancer cells and the replication of viral pathogens. The ability of nucleoside analogues to interfere selectively with nucleotide and nucleic acid biosyntheses makes these compounds ideal anti-cancer and anti-viral agents. Nucleoside analogues inhibit the synthesis of nucleotides and nucleic acids by competing with their naturally occurring counterparts for the binding sites on enzymes that are involved in nucleic acid biosynthesis (Perigaud et al., Nucleosides and Nucleotides, 11:903 (1992)). Consequently, the formation of nucleic acid precursors, including nucleosides and nucleotides, is prevented. Furthermore, the incorporation of the analogues into nucleic acid molecules hinders the replication and/or transcription of these molecules. Therefore, the administration of nucleoside analogues to cancerous cells can inhibit their proliferation, whereas the administration of such analogues to virally infected cells can inhibit the replication of the infecting virus.
Because they are relatively small in size and electrostatically neutral, nucleoside analogues readily traverse the plasma membrane, facilitating passage to their intracellular target sites. However, the therapeutic effect of nucleoside analogues is severely limited by their dependence on intracellular enzymes for conversion into active inhibitors. The attachment of phosphate groups to the sugar residues of the nucleoside analogues is a necessary step for inhibiting nucleotide and nucleic acid biosynthesis. In some cases, nucleoside analogues must undergo two or more steps for conversion into active metabolites, thereby increasing the time it takes for them to act as inhibitors and the chance that the analogues will be converted to inactive metabolites, ultimately decreasing their effectiveness.
The dependence on intracellular enzymes for conversion into activating inhibitors is alleviated through the use of nucleotide analogues, which contain the phosphate groups. Although they offer a more direct mode of action in comparison to nucleoside analogues, the delivery of nucleotide analogues to the interior of a cell is severely hindered by the highly negative charge of the phosphate group. Therefore, a mechanism by which nucleotide analogues are delivered to their intracellular targets is desired.
Gmeiner et al. (U.S. Pat. No. 5,457,187) discloses the delivery of the nucleotide analogue, 5-fluorodeoxyuridine 5′ monophosphate, through covalent attachment of lipophilic or cationic moieties, including cholesterol, ethyl-spaced adamantane, 1,2-dihexadecylglycerol and poly-L-lysine. The entry into cells is further enhanced by delivering the analogue as a homo-oligomer. Although these conjugates provide a mechanism of drug delivery to the interior of the cell, they do not target the nucleotide analogue to specific tissues or cells.
As with most drugs, the clinical application of nucleotide analogues is limited by their failure to reach the targeted cell population and by the toxicity they impose on non-targeted cells. Tissue-specific drug targeting would not only reduce systemic toxicity but would also potentiate drug action by concentrating the drug in target cells or tissues (Wadhwa et al., J. Drug Targeting 3:111 (1995)). Targeting nucleotide analogues in a tissue-specific manner is, therefore, desired.
Several tissue-specific drug targeting strategies have consequently come about in an attempt to overcome these problems. One such strategy, carrier-mediated drug targeting, involves either the covalent or non-covalent association of a drug with a tissue-specific targeting moiety. In a subclass of this strategy, termed active targeting, the targeting moiety is a ligand that is recognized by a specific receptor, which is found predominantly at the target site. Ligand binding to the receptor results in receptor-mediated endocytosis, wherein the drug-ligand conjugate is internalized, along with the receptor, by the target cell. Once inside the cell, the conjugate is susceptible to intracellular enzymes that cleave the bond between the ligand and drug, resulting in the release of the drug from the conjugate. In this manner, the delivery of therapeutic agents to targeted cell populations is achieved.
Ligand-directed, receptor-mediated endocytosis is the basis upon which several drug targeting systems rely. Antibodies, or fragments thereof, hormones, cytokines, and other soluble proteins, such as gastrin and transferrin, have all been employed as targeting ligands for the delivery of drugs to specific cell populations. For example, Myers et al. (U.S. Pat. No. 5,087,616) discloses a drug delivery conjugate wherein the drug, daunomycin, is delivered to epithelial cells specifically via conjugation to the hormone ligand epithelial growth factor. Also, Gmeiner et al. (U.S. Pat. No. 5,663,321) discloses the delivery of a homo-oligomer comprising monomers of the nucleotide analogue, 5-fluorodeoxyuridine 5′ monophosphate, through attachment to an antibody or fragment thereof.
Ligands are not always protein in nature, however. Carbohydrate moieties serve as the ligands for a family of receptors known as lectins. These receptors vary, based on their tissue expression and ligand specificity. For instance, the lectin found on Kupffer cells is specific for mannose, whereas the lectin found on hepatic endothelial cells binds selectively to fucose residues.
The asialoglycoprotein receptor (ASGPR), a lectin found predominantly on the surface of hepatocytes, has been studied in depth for the purpose of delivering therapeutic agents to the liver. Because it mediates high affinity interactions with practically any entity that contains terminal galactose or acetylgalactosamine residues, irrespective of the size or structure of the entity, the ASGPR is a model system to use for drug delivery. Pioneering work by Wu et al. (J. Biol. Chem. 262:4429 (1987)) demonstrated that antisense oligonucleotides electrostatically complexed to poly-L-lysine that is linked to asialoorosomucoid, a galactose/acetylgalactosamine-containing protein, are efficiently and specifically taken into human hepatocellular carcinoma cells through direct interaction with the ASGPR. However, because the antisense oligonucleotides were non-covalently attached to the targeting moiety, it is reasonable to assume that the resulting conjugate was less biostable than if it were covalently bonded.
Tissue-specific drug targeting through use of the ASGPR has been applied to the delivery of nucleotide analogues. For instance, Groman et al. (U.S. Pat. No. 5,554,386) discloses a conjugate comprising the nucleotide analogue, araAMP, and the polysaccharide, arabinogalactan, which binds to the ASGPR, for delivery of the drug to hepatocytes. Because polysaccharides contain multiple drug attachment sites, however, the synthesis of this conjugate would result in a heterogeneous mixture of conjugates, wherein the number of nucleotide analogues per conjugate varies from batch to batch. Consequently, the IC50 of the conjugate would also vary from batch to batch, making it very difficult to determine the dose for administration.
Rohlff et al. (Cancer Research 59:1268 (1999)) discloses a drug-ligand conjugate, OGT719, wherein a single galactose moiety is covalently attached to the nucleoside analogue, 5-fluorouracil. Although shown to be effective in animal models, this drug conjugate is predicted to bind weakly to the ASGPR in view of the studies conducted by Lee et al. (Biochemistry 23: 4255 (1984)), in which ligands containing three or four galactose residues arranged in a specific conformation bind with much greater affinity to the ASGPR than a single residue. High affinity binding conjugates are desired so that lower doses of the conjugates can be administered for achievement of the desired therapeutic effects.
Taken together, a conjugate capable of undergoing ligand-directed, receptor-mediated endocytosis for the delivery of nucleotide analogues to their intracellular targets in a tissue-specific manner is desired. The components that comprise the conjugate should be covalently bonded to each other for maximum biostability. The conjugate should be chemically defined and structurally homogeneous such that a single IC50 can be determined. The conjugate also should bind with high affinity to a targeted receptor. A conjugate with these properties would have an improved therapeutic index. It is an object of the present invention to provide such a conjugate. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein.